<i>Candida rugosa</i> Lipase Bioconjugation to Cellulose Nanocrystals with High Immobilization Efficiency: Comparison with Nonspecific Approach

Nanostructured materials are promising substrates for biocatalyst immobilization. We report a green and sustainable strategy for enzyme immobilization using cellulose nanocrystals (CNCs) derived from renewable sources. CNCs offer biodegradability, low toxicity, and high surface area, enabling efficient immobilization of <i>Candida rugosa</i> lipase (<i>CRL</i>). Covalent bioconjugation on TEMPO-oxidized cellulose nanocrystals (TO_CNCs) provides an almost quantitative immobilization yield without releasing toxic byproducts, but with reduced enzymatic activity per mg of immobilized protein. Conversely, nonspecific immobilization on sulfated cellulose nanocrystals (S_CNCs) shows very low immobilization yield but preserves enzyme mobility and slightly enhances activity. The immobilized biocatalysts were characterized by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, high-resolution synchrotron X-ray diffractometry (XRD), ultraviolet-visible spectroscopy (UV-vis), field emission scanning electron microscopy (FE-SEM), bicinchoninic acid assay (BCA), solid-state nuclear magnetic resonance (ssNMR) spectroscopy, and enzymatic activity measurements. Notably, ssNMR reveals the effectiveness of TO_CNCs in preventing enzyme dispersion.